Seipin is a discrete homooligomer

Abstract

Seipin is a transmembrane protein that resides in the endoplasmic reticulum and concentrates at junctions between the ER and cytosolic lipid droplets. Mutations in the human seipin gene, including the missense mutation A212P, lead to congenital generalized lipodystrophy (CGL), characterized by the lack of normal adipose tissue and accumulation of fat in liver and muscles. In both yeast and CGL patient fibroblasts, seipin is required for normal lipid droplet morphology; in its absence droplets appear to bud abnormally from the ER. Here we report the first purification and physical characterization of seipin. Yeast seipin is in a large discrete protein complex. Affinity purification demonstrated that seipin is the main if not exclusive protein in the complex. Detergent sucrose gradients in H(2)O, and D(2)O and gel filtration were used to determine the size of the seipin complex and account for detergent binding. Both seipin-myc13 (seipin fused to 13 tandem copies of the myc epitope) expressed from the endogenous promoter and overexpressed seipin-mCherry form ∼500 kDa proteins consisting of about 9 copies of seipin. The yeast orthologue of the human A212P allele forms only smaller complexes and is unstable; we hypothesize that this accounts for its null phenotype in humans. Seipin appears as a toroid by negative staining electron microscopy. We speculate that seipin plays at least a structural role in organizing droplets or in communication between droplets and ER.

Figure 1

Wild-type and mutant seipins migrate as discrete oligomers. (A) Immunoblots of fractions from detergent glycerol gradients. Columns indicate wild-type or G225P seipin sequence, rows indicate the carboxy-terminal tag of the fusion protein, which is driven either by the PGK1 promoter on a plasmid or by the endogenous chromsosomal seipin promoter, as indicated by parentheses. For each blot the positions of the pellet, first, and twentieth fraction (from bottom to top of gradient) are indicated. Arrowheads denote the position of internal native standards as determined by densitometry of Amido Black-stained nitrocellulose used later for the immunoblots. The standards are as follows: t, thyroglobulin (669 kDa); c, catalase (232 kDa), not included for mCherry constructs due to cross-reactivity with the antibody; l, lactate dehydrogenase (140 kDa); and a, albumin (67 kDa). The nitrocellulose was immunoblotted for mCherry (first row), seipin (second row), or myc (third and fourth rows). Prestained molecular mass markers (kDa) are shown to the left of panels. (B) Seipin[G225P] is unstable. Cells overexpressing untagged wild-type or seipin[G225P] were subjected to cycloheximide (CHX) in the presence of the proteosomal inhibitor MG132 or its vehicle DMSO. Cells were lysed at the times indicated after addition of CHX and subjected to SDS–PAGE and immunoblotting with anti-seipin antibody. Arrow, seipin; asterisk, cross-reacting band.

Figure 2

Hydrodynamic behavior of seipin-mCherry and seipin[G225P]-mCherry. (A) Rows 1 and 2: Immunoblots of fractions from detergent H₂O– or D₂O–sucrose gradients of seipin-mCherry (“wild-type” column) and seipin[G225P]-mCherry (“G225P” column). See legend to Figure 1 for more details. Arrow indicates the G225P form, which is difficult to visualize. Row 3: Immunoblots of fractions from S500 chromatography. White arrowheads on each blot indicate void volume, left, and column volume, right. Native markers identical to Figure 1 except b, 5 nm nanobeads. (B) Curve of Stokes radii of standards from S500, with arrow indicating behavior of seipin[G225P]-mCherry (“G225P”).

Figure 3

Hydrodynamic behavior of seipin-myc13 and seipin[G225P]-myc13. Identical to Figure 2 except seipins were tagged with 13 tandem copies of the myc epitope (in the chromosome to use the endogenous promoter) instead of overexpressing seipin-mCherry forms on plasmids, and we used 70S E. coli ribosomes “r” instead of beads as a marker. “*”, much of this fraction was lost during processing.

Figure 4

Seipin can self-associate. Anti-myc immunoblots from detergent glycerol gradients. Besides expressing seipin-myc13 from the endogenous chromosomal promoter, cells also oveexpressed either seipin-mCherry (top row) or seipin[G225P]-mCherry (bottom row). See legend to Figure 1 for more details. Note the shift in the complex to lower MW when coexpressed with seipin[G225P]-mCherry.

Figure 5

Seipin is a homooligomer. ER-enriched membranes from wild type (lanes 1, 3, and 5) or the seipin-myc13-overexpressing strain (lanes 2, 4, and 6) were solubilized in detergent and incubated with anti-myc epitope antibody-conjugated beads. Protein was eluted with elution buffer (from manufacturer) (lanes 1 and 2) or eluted with TEV protease (lanes 3–6). Protease was removed on a nickel column (lanes 5 and 6). Samples were subjected to SDS–PAGE and stained with silver (A) or immunoblotted with anti-seipin antibody (B). The migration of the seipin-myc_x ladder, seipin, and TEV protease are indicated in (A).

Figure 6

Seipin forms a toroid. Negative staining of (A) affinity-purified seipin-myc13 from overexpressing cells, eluted from antibody column with elution buffer from the manufacturer. (B and C) Affinity-purified seipin-TEVsite-myc13 (C) eluted with TEV protease or a parallel sample from nonexpressing cells (B).